| Environmental agents are involved in the initiation and promotion of numerous human cancers. Despite the diversity of their structures and reactivities, the unifying principle of environmental carcinogenesis is the environmental agents' ability to interact with cellular DNA, thereby producing mutations and heritable changes. Ultraviolet radiation (UV) is the most common physical carcinogen in our environment, and has been identified as a major etiological factor in the development of skin cancer in humans.;Immunologic assays have been developed for quantitation of photoproducts in DNA extracted from UV exposed tissues. The efficacy of these assays for use in molecular epidemiological risk assessment is highly dependent upon the affinity and specificity of the monoclonal antibodies used for DNA adduct determination. However, hybridoma production is a time-consuming and costly process, which may not yield an antibody with the desired specificity and affinity. In addition, in vivo selective pressures make it difficult to produce auto-antibodies, such as those against DNA adducts. The stochastic nature of monoclonal antibody production has been a major frustration and impediment for the implementation of many biomonitoring investigations. The union of molecular biology and immunology has yielded a powerful technology that may allow antibodies to be generated on demand, without reliance on the vagaries of animal based production.;This thesis study examines the efficacy of combining phage display and molecular biological techniques to produce antibodies with altered affinities and specificities in vitro. The alpha-UVssDNA-1 monoclonal antibody, produced by murine hybridoma 25JF.C3B6, has been used to detect pyrimidine-pyrimidone (6-4) thymine-thymine (T[6-4]T) photoproducts in cells and tissue. However, the usefulness of this antibody is limited because of low specificity. A single chain variable fragment (ScFv) was constructed from 25JF.C3B6 hybridoma mRNA and displayed on the surface of filamentous bacteriophage. The recombinant bacteriophage were affinity selected against UV irradiated polydeoxythymidylic acid, and clones which specifically recognized T(6-4)T DNA adducts were isolated. Soluble ScFv molecules were affinity purified and the binding characteristics of the antibody fragment compared to those of the native immunoglobulin. Affinity of the soluble antibody fragments appear to be reduced compared to the alphaUVssDNA-1 monoclonal antibody, however the reduction may be due to various factors, including instability of the ScFv molecule. |
